Korean Journal of Cerebrovascular Surgery 2008;10(3):437-441.
Published online September 1, 2008.
Time- dependent Changes of the Infarct Volume in a Rat Stroke Model: A Comparison of the Use of MRI and TTC- staining as Monitoring Tools.
Kwon, Ji Woong , Park, Chul Kee , Shin, Hye Young , Paek, Sun Ha , Wang, Kyu Chang , Kim, Dong Gyu
Department of Neurosurgery, Seoul National University College of Medicine, Clinical Research Institute, Seoul National University Hospital, Seoul Korea. paeksh@snu.ac.kr
Abstract
OBJECTIVE
S: Serial changes of focal ischemic lesions as seen on magnetic resonance (MR) images and triphenyltetrazolium chloride (TTC)-stained samples of transient middle cerebral artery occlusion in a rat model were evaluated to investigate the natural course of the lesions and the feasibility of the use of each method as a monitoring tool. METHODS: Transient middle cerebral artery occlusion (MCAO) was induced in fifteen adult female Sprague Dawley rats using the method of intraluminal vascular occlusion. Two hours after MCAO was induced, reperfusion was performed. Serial MR images were obtained and the volume of the brain infarct was estimated. For macroscopic and microscopic evaluation of the ischemic lesions, the ten animals were sacrificed at different times after MCAO. The rat brains were then removed and six coronal sections were made. Each section was incubated at 37 degrees C in 2% TTC solution for 15 minutes. RESULTS: Postischemic injury evaluations that were made periodically for eight weeks revealed that the lesion volume as determined from T2 maps had reached a peak on the second day after ischemic injury and the volume decreased afterwards for one week; by the fourth week, the lesion volume again increased to stabilize initial lesion development. There were considerable discrepancies between the infarct area of the samples determined by TTC staining and the in vivo infarct area estimated from the MR images, especially for late stages. CONCLUSION: T2 map MR images, with a careful consideration of the natural course of infarction development, can provide an adequate and noninvasive means to evaluate the degree of ischemic injury under diverse experimental circumstances.
Key Words: Rat stroke model, Infarct volume, MRI, TTC staining

Introduction


  
Animal models of stroke remain essential for understanding the mechanisms underlying brain tissue damage and the development of therapeutic strategies. Because of its noninvasive nature, its versatility, and its potential for imaging at relatively high spatial and temporal resolution, magnetic resonance (MR) imaging has evolved into a promising tool for biomedical experiments.8) In adult rodents, MR images have been demonstrated as a useful method in the evaluation of acute ischemic studies as well as for the follow-up of stroke evolution.10)12)18) However, reported experiences of MR images for ischemic tissue injury were time-limited results, as only four week-long observation have been previously described.10)11)13)24) Recently, the need for suitable methods for the long-term evaluation of experimental ischemic changes in the rodent central nervous system has been raised because of the evolution of cell therapy strategy in which experiments need a more significant amount of time for evaluation. Although 2,3,5- triphenyltetrazolium chloride (TTC) staining is the most common method for differentiating viable tissue from infarcted tissue macroscopically, it is also questionable whether TTC staining can provide appropriate serial quantitative data of the long-term effects of ischemic tissue injury.5) The objective of this paper is studying serial change of focal ischemic lesions in MR images and TTC-stained samples to investigate their natural course and feasibility as a monitoring tool for the purpose of more appropriate application of these methods to future experiments. 

Materials and Methods 

Materials 
   Fifteen adult female Sprague Dawley rats weighing 250-300 g were used. Animals were housed at a controlled temperature (21±1℃) with a 12 h light-dark cycle. Standard laboratory rat food pellets and tap water were supplied ad libitum. 

Induction of stroke 
   Rats were anesthetized with an intraperitoneal injection of 1% ketamine (30mg/kg) and xylazine hydrochloride (4mg/kg). Their rectal temperature was controlled at 37℃ with heat blanket. 
   Transient middle cerebral artery occlusion (MCAO) was induced using a method of intraluminal vascular occlusion described elsewhere.14) Briefly, a 2cm incision was made at the center of the neck, and the left common carotid artery, external carotid artery, and internal carotid artery (ICA) were exposed under an operating microscope. A length of 3/0 monofilament nylon suture with its tip rounded by heating near a flame was advanced into the lumen of the ICA until it blocked the origin of the middle cerebral artery. Two hours after MCAO, animals were reanesthetized, and reperfusion was performed by withdrawal of the suture until its tip reached the insertion point. Free access to food and water was allowed after recovery from anesthesia. 

MR imaging 
   Serial MR images were taken daily from the day of MCAO to the fourth day after MCAO in one rat. Follow-up MR imaging was performed every week for eight weeks after MCAO in four rats. In ten rats, the MR imagings were taken weekly until they were killed. Rats were anesthetized with the intraperitoneal injection of 1% ketamine (30mg/kg) and xylazine hydrochloride (4mg/kg). A 1.5 T Advantage Horizon System (GE, Milwaukee, WI, USA) equipped with an actively shielded gradient coil, and wrist coil (GE), was used in this study. A solenoid-type volume coil 70mm in diameter for transmission and a surface coil 22mm in diameter for signal detection were combined perpendicularly. T2-weighted axial images were acquired with 3,500 ms repetition time (TR), 85 ms echo time (TE), 6 mm field of view (FOV), 256×160 resolution, and 2mm slice thickness at the center of the brain in the axial plane. Two-dimensional gradient-echo axial images were acquired with 300ms TR, 20ms TE, 20 degrees of flip angle, 60mm FOV, and 2 mm slice thickness at the center of the brain. 

TTC staining 
   For the macroscopic and microscopic evaluation of ischemic lesions, the ten animals were killed two at a time in two days, and one, two, four, and eight weeks after MCAO. The rats were euthanized with an overdose of pentobarbital after MR imaging follow-up. Brains were then removed and six coronal sections (2mm thick slices from anterior 3.5mm to anterior 13.5mm) were made using a brain matrix (Agar Scientific, UK). Each section was incubated at 37℃ in 2% TTC solution for 15min. Then normal brain tissue stained brick red, while the area of infarct remained pale. The sections were then fixed with 10% formalin and processed with hematoxylin-eosin staining for microscopic examination. 

Mesurements of the infarct volume 
   Morphometric measurements of the infarct volume of the rat brain were made using noncommercial software (Osiris, version 4.0). The volume of the brain infarct was estimated by measuring the volume of the high signal area ipsilateral to the MCAO in T2-weighted images and the pale area in TTCstained brain sections. 

Results 

Infarct volume in MR images 
   Infarct volume changes in MR images showed same trends with time in all rats. During the first week after MCA occlusion, the measured infarct volume in T2 maps emerged on the day of injury and reached a maximum in the second day(Fig. 1). From then on, infarct volume changes in T2 maps were expressed as a ratio of the volume measured weekly to the volume on the second day. Infarct volume continued to decrease after the second day of MCA occlusion until the first week and then gradually increased and reached the plateau after a month(Fig. 2). 

Infarct volume in TTC stained samples 
   Measured infarct volumes in T2 maps were well correlated with the ischemic lesion seen by TTC staining during the first week. However, appropriate measurement of ischemic lesion volume could not be executed with samples obtained after one week, owing to the tissue loss at the infarct area(Fig. 3). Histological examination of lesion area in TTC staining confirmed the infarction. 

Discussion 

   Within an ischemic lesion, MR parameters are timedependent and spatially heterogeneous.23) Many attempts have been tried with diverse sequences of MR images to monitor ischemic lesions in rodents for diagnostic assessments, tissue characterization, prognosis prediction, and estimation of therapeutic response.2)4)7)11)18)19)23) T2 map MR images have long been considered as the gold standard measurement to identify ischemic lesions.11) Although inaccuracy in the estimation of infarct volume is indicated during the early phase, T2 map MR images are known to provide the most consistent measurement corresponding with final lesion size.10) It has been demonstrated that the size of the infarct does not increase further after 24 hours of ischemia, so the infarct volume on T2 maps, 24 hours after occlusion, can be considered as the final size.21) Our result demonstrated that the lesion volume in T2 maps reached a peak on the second day after ischemic injury and decreased afterwards for up to one week after injury, and then increased again to stabilize by the fourth week after initial lesion development. The increase in T2 abnormality during the first two days may reflect an increase of brain edema.10) Therefore, there is a possibility of overestimation of infarct volume in T2 maps during the early phase, even though the discrepancy may be insignificant when compared with that of TTC staining results shown in the present study. 
   Derugin et al.7) suggested the usefulness of T2 and diffusion-weighted maps taken one week after temporary ischemic injury in neonatal rat brain as a surrogate measure of the long-term endpoint of stroke studies. Jiang et al.11) showed significant correlations between MR image signatures at different time points and histopathological measurements of lesion area obtained at one week. We also observed a similar correspondence between T2 map lesions and TTC staining area one week after ischemic injury. However, infarct volume measured in T2 maps were then smaller than that of the final lesions, which could be attained at four weeks after injury. This result is inconsistent with previous rodent studies.10)24) Virley et al.24) showed in their MR images that infarct size in rats remained constant from seven to 28 days, by which time lesion development was considered to be complete. In a primate study, however, the phenomenon of late increase of infarct volume in MR images had been reported.15) Although the reason for these late changes is obscure, it can be partially explained by the MRI fogging phenomenon observed in human studies.1)22) During the early phase of stroke, magnetically active erythrocytes leaking out of pathologically altered blood vessels, the replacement of damaged tissue with scavenging, lipid-laden macrophages and a decrease in bulk water may lead to masking of the true extent of the lesion when examined using MRI in which lipid-filled macrophages would displace water, reducing the size of damage detected on the T2 map images.15)22) When the number of macrophages has returned to normal, the true extent of the lesion would again become apparent on T2W images.15) This fogging effect and the four-stage pathological process of neuronal death, inflammation, reorganization, and resolution that is thought to be complete by 30th days in rats can account for the course of infarct volume measured in T2 maps as shown in the present study.6) 
   Our result also demonstrated that there is a problem with the TTC staining method for volumetric analysis of infarct with time. There are considerable discrepancies between the infarct samples with TTC staining and in vivo infarct area estimated by MR images. Apart from the technical tissue loss problems in infarct area during the process, the measurement of the volume of ischemia by TTC staining disclosed many points of uncertainty.3) Many papers have indicated false positive or false negative detection of the infarction area by TTC staining owing to transient inactivation of enzymatic activities or to different staining methods.9)16)17)20) These potential pitfalls cast some doubt on the validity of TTC staining as a reliable method for analysis of infarct volumes and imply that the results should be interpreted more cautiously. 

Conclusion 

   Periodic evaluation for eight weeks after ischemic injury revealed that the lesion volume in T2 maps reached a peak on the second day after ischemic injury and decreased afterwards for one week, and then increased again to stabilize by the fourth week initial lesion development. There were considerable discrepancies between the infarct samples with TTC staining and in vivo infarct area estimated by MR images, especially in the late stages. Therefore, T2 map MR images, with careful consideration of the natural course of infarction development, can provide an adequate and noninvasive method to evaluate the degree of ischemic injury in diverse experimental circumstances. 


REFERENCES


  1. Asato R, Okumura R, Konishi J. "Fogging effect" in MR of cerebral infarct. J Comput Assist Tomogr 15:160-2, 1991 

  2. Barbier EL, Liu L, Grillon E, Payen JF, Lebas JF, Segebarth C, et al. Focal brain ischemia in rat: acute changes in brain tissue T1 reflect acute increase in brain tissue water content. NMR Biomed 18:499-506, 2005 

  3. Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing LG Jr, et al. Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res 1116:159-65, 2006 

  4. Chen F, Suzuki Y, Nagai N, Peeters R, Marchal G, Ni Y. Dynamic susceptibility contrast-enhanced perfusion MR imaging at 1.5 T predicts final infarct size in a rat stroke model. J Neurosci Methods 141:55-60, 2005 

  5. Chiamulera C, Terron A, Reggiani A, Cristofori P. Qualitative and quantitative analysis of the progressive cerebral damage after middle cerebral artery occlusion in mice. Brain Res 606:251-8, 1993 

  6. Clark RK, Lee EV, Fish CJ, White RF, Price WJ, Jonak ZL, et al. Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study. Brain Res Bull 31:565-72, 1993 

  7. Derugin N, Dingman A, Wendland MF, Fox C, Bollen A, Vexler ZS. Magnetic resonance imaging as a surrogate measure for histological sub-chronic endpoint in a neonatal rat stroke model. Brain Res 1066:49-56, 2005 

  8. Dijkhuizen RM, Nicolay K. Magnetic resonance imaging in experimental models of brain disorders. J Cereb Blood Flow Metab 23:1383-402, 2003 

  9. Duckworth EA, Butler TL, De Mesquita D, Collier SN, Collier L, Pennypacker KR. Temporary focal ischemia in the mouse: technical aspects and patterns of Fluoro-Jade evident neurodegeneration. Brain Res 1042:29-36, 2005 

  10. Hofmeijer J, Veldhuis WB, Schepers J, Nicolay K, Kappelle LJ, Bar PR, et al. The time course of ischemic damage and cerebral perfusion in a rat model of space-occupying cerebral infarction. Brain Res 1013:74-82, 2004 

  11. Jiang Q, Chopp M, Zhang ZG, Knight RA, Jacobs M, Indham JP, et al. The temporal evolution of MRI tissue signatures after transient middle cerebral artery occlusion in rat. J Neurol Sci 145:15-23, 1997 

  12. Jung JM, Chung YS, Park IS, Lee SH, Kim HJ, Choi KS, et al. The time evolution of cerebral infarction in rat middle cerebral artery occlusion. J Korean Neurosurg Soc 22:404-12, 1993 

  13. Kovacs Z, Ikezaki K, Takahashi M, Kawai J, Fukui M. A chronological evaluation of experimental brain infarct by diffusion-mapping and magnetization transfer contrast imaging. Acta Neurochir Suppl 70:43-5, 1997 

  14. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84-91, 1989 

  15. Marshall JW, Ridley RM, Baker HF, Hall LD, Carpenter TA, Wood NI. Serial MRI, functional recovery, and long-term infarct maturation in a non-human primate model of stroke. Brain Res Bull 61:577-85, 2003 

  16. Marshall RS, Lazar RM, Pile-Spellman J, Young WL, Duong DH, Joshi S, et al. Recovery of brain function during induced cerebral hypoperfusion. Brain 124:1208-17, 2001 

  17. Memezawa H, Smith ML, Siesjo BK. Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke 23:552-9, 1992 

  18. Meng X, Fisher M, Shen Q, Sotak CH, Duong TQ. Characterizing the diffusion/perfusion mismatch in experimental focal cerebral ischemia. Ann Neurol 55:207-12, 2004 

  19. Ono Y, Morikawa S, Inubushi T, Shimizu H, Yoshimoto T. T2*-weighted magnetic resonance imaging of cerebrovascular reactivity in rat reversible focal cerebral ischemia. Brain Res 744:207-15, 1997 

  20. Park CK, Mendelow AD, Graham DI, McCulloch J, Teasdale GM. Correlation of triphenyltetrazolium chloride perfusion staining with conventional neurohistology in the detection of early brain ischaemia. Neuropathol Appl Neurobiol 14:289-98, 1988 

  21. Quast MJ, Huang NC, Hillman GR, Kent TA. The evolution of acute stroke recorded by multimodal magnetic resonance imaging. Magn Reson Imaging 11:465-71, 1993 

  22. Scuotto A, Cappabianca S, Melone MB, Puoti G. MRI "fogging" in cerebellar ischaemia: case report. Neuroradiology 39:785-7, 1997 

  23. Soltanian-Zadeh H, Pasnoor M, Hammoud R, Jacobs MA, Patel SC, Mitsias PD, et al. MRI tissue characterization of experimental cerebral ischemia in rat. J Magn Reson Imaging 17:398-409, 2003 

  24. Virley D, Beech JS, Smart SC, Williams SC, Hodges H, Hunter AJ. A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior. J Cereb Blood Flow Metab 20:563-82, 2000

TOOLS
METRICS Graph View
  • 666 View
  • 7 Download
Related articles


ABOUT
BROWSE ARTICLES
EDITORIAL POLICY
FOR CONTRIBUTORS
Editorial Office
The Journal of Cerebrovascular and Endovascular Neurosurgery (JCEN), Department of Neurosurgery, Wonkwang University
School of Medicine and Hospital, 895, Muwang-ro, Iksan-si, Jeollabuk-do 54538, Korea
Tel: +82-2-2279-9560    Fax: +82-2-2279-9561    E-mail: editor.jcen@the-jcen.org                

Copyright © 2024 by Korean Society of Cerebrovascular Surgeons and Korean NeuroEndovascular Society.

Developed in M2PI

Close layer
prev next